Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.

Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and -4C.

Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography. The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (> 95%) as estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4 are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in cells.

Characterization of the rolipram-sensitive, cyclic AMP-specific phosphodiesterases: identification and differential expression of immunologically distinct forms in the rat brain.

To determine the properties of the cAMP-specific, rolipram-sensitive phosphodiesterases (cAMP-PDEs) that are expressed in different organs, monoclonal and polyclonal antibodies were raised against different epitopes present in the cAMP-PDE sequences. Of the several antibodies generated against peptides and fusion proteins, one monoclonal and four polyclonal antibodies recognized both the native cAMP-PDEs as well as the denatured proteins on Western immunoblot analysis. An immunoprecipitation assay demonstrated that these antibodies recognized the recombinant rat PDE4A, PDE4B, and PDE4D proteins with different avidity. The polyclonal antibody K118 and the monoclonal M3S1 were most specific for rat PDE4B and PDE4D forms, respectively, whereas the AC55 antiserum displayed the highest affinity for PDE4A forms. This selectivity was confirmed by Western blot analysis using recombinant rat PDE4A, PDE4B, and PDE4D proteins expressed in a heterologous system. These antibodies were used to characterize the cAMP-PDEs expressed in the rat brain. An immunoblot of extract of cortex and cerebellum demonstrated that at least seven different polypeptides specifically cross-reacted with the different antibodies, indicating that multiple cAMP-PDEs are expressed in this tissue. On the basis of cross-reactivity with PDE4D but not PDE4A or PDE4B antibodies, 93- and 105-kDa PDE4D species were detected in the cortex and cerebellum extract. These forms are different from the 68-kDa PDE4D form expressed in endocrine cells after hormonal stimulation. Although the 93-kDa form was recovered in both the soluble and particulate fractions, the 105-kDa polypeptide was mostly particulate in the cortex and cerebellum extracts. PDE4B forms of 90-87 kDa were recovered in both soluble and particulate compartments of the brain extract. These forms were different from the previously identified PDE4A variants of 110 and 75 kDa. These data demonstrate that the presence of multiple cAMP-PDE genes is translated into cAMP-PDE proteins of different sizes and distinct immunological properties and that multiple variants derived from these cAMP-PDE genes are expressed in different regions of the brain and different subcellular compartments. These immunological tools will be useful to identify different cAMP-PDE forms expressed in organs targeted for pharmacological intervention with PDE4 inhibitors.

Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis.

Four closely related cyclic-nucleotide specific phosphodiesterase (PDE4) genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame (ORF) of 791 amino acids (aa) is encoded by PDE4C-791, which is similar to a recently described cDNA [Engels, P., Sullivan, M., Muller, T. and Lubbert, H. FEBS Lett. 358 (1995) 305-10], except that an alternative 5'-end sequence upstream of the first methionine extends the PDE4C-791 ORF by 79 aa. The PDE4C-426 variant contains 3 insertions that are located 5' to the catalytic domain and encode several in-frame stop codons. The predicted 426 aa protein initiates at a methionine 365 aa within PDE4C-791. A baculovirus clone starting at this methionine expressed an enzymatically active protein. Two additional splice variants, PDE4C-delta54 and PDE4C-delta109, were found in testis mRNA. PDE4C-delta54 contained a novel 5'-end region and a deletion of 162 nt; the predicted protein deletes 54 aa from the amino-terminal region. The PDE4C-delta54 protein produced in baculovirus-infected cells was enzymatically active and sensitive to PDE4-specific inhibitors. The PDE4C-delta109 protein is similar to PDE4C-delta54 but has an additional 55 aa deleted in the catalytic domain; it lacked enzymatic activity. Analysis of uncloned total mRNA from 4 tissue sources by polymerase chain reaction (PCR) confirmed the presence of mRNAs with the two deletions and three insertions that we observed in cDNA clones. The PDE4C-delta54 variant was found only in testis and the 5'-extended region of PDE4C-791 was seen only in lung and the melanoma cell line G361. Hence, tissue-specific expression of various PDE4C isoforms should be considered in understanding how these gene products modulate cellular responses to cAMP.

Activation and selective inhibition of a cyclic AMP-specific phosphodiesterase, PDE-4D3.

Prostaglandin E2 produces a transient increase in the intracellular concentration of cAMP in a human promonocytic cell line (U937). The temporal pattern consists of a rapid increase followed by a gradual decline to a new steady state. The decline phase coincides with an increase in the activity of a high affinity form of cAMP phosphodiesterase (PDE). Immunoprecipitation with specific antibodies revealed that the activated enzyme is a variant of PDE-4D. To confirm this observation, three isoforms of human PDE-4 (A, B, and D) were cloned and expressed in Sf9 cells with recombinant baculovirus infection. The activity of only one of the isoforms (PDE-4D3) increased after incubation with the catalytic subunit of protein kinase A and Mg-ATP. Hydrolytic activity of human PDE-4D3 was dependent on Mg2+. Before phosphorylation, the concentration-response curve for Mg2+ was biphasic and ranged from 0.1 to 100 mM. Phosphorylation of PDE-4D3 by protein kinase A produced a monophasic Mg2+ response curve (0.5 Vmax = 0.2 mM). Phosphorylation of PDE-4D3 increased the sensitivity of the enzyme to inhibition by RS-25344 (approximately 100-fold) and RS-33793 (approximately 330-fold). Thus, phosphorylation of PDE-4D3 induces an apparent conformation change that increases maximum velocity and sensitivity to inhibition by some analogues of nitraquazone. These observations provide the basis for a novel pharmacological strategy that targets an activated form of PDE in human leukocytes. Selective PDE-4D3 inhibitors may have useful anti-inflammatory properties with fewer adverse side effects than other PDE-4 inhibitors.

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain.

Alzheimer's disease (AD) is characterized by the extensive deposition of the 42-amino-acid beta-amyloid or A4 protein in neuritic plaques and neurofibrillary tangles within the brain. This protein is liberated from the much larger amyloid protein precursor (APP). Multiple species of APP have been proposed, including several forms that contain a 56 amino acid insert sequence analogous to the Kunitz protease inhibitors. Although expression of APP mRNA is reportedly altered in AD brain and various roles for APP have been proposed, the pathogenesis of amyloid deposition and AD remains unclear. AD is also characterized by specific memory impairments associated with decreased cholinergic activity. While aging rats do not develop mature amyloid pathology, behaviorally impaired aged rats demonstrate an analogous cholinergic decline. In this study, we examined behaviorally characterized aged rats and normal young controls for changes in APP immunohistochemistry by using anti-APP antibodies, which detect N- or C-terminal regions and which distinguish APP species with or without the Kunitz protease inhibitor domain. The results show specific age- and behavior-related changes in cortical APP immunoreactivity as well as limited numbers of APP immunoreactive deposits in the aged rats. Additionally, we found that lesions of the fimbria-fornix pathway, which in part mimic the memory impairments and loss of cholinergic activity seen in AD, result in the marked accumulation of APP immunoreactive material in the region of cholinergic fiber degeneration in the hippocampus. These findings are discussed in relation to the pathogenesis of AD in humans.

Differential distribution of amyloid protein precursor immunoreactivity in the rat brain studied by using five different antibodies.

The beta-amyloid or A4 protein is found deposited in neuritic plaques and neurofibrillary tangles in Alzheimer's disease (AD) affected brains and in the brains of adults with Down's Syndrome. The precursor to this 42 amino acid protein is the 695 amino acid long amyloid protein precursor (APP-695). Two additional APP species, APP-751 and APP-770, each contain a 56-amino-acid insert sequence that is analogous to Kunitz protease inhibitors. APP mRNA is widely distributed in both the human and rat brain, although the adult rat does not develop mature amyloid pathology. In this study we used antibodies against the N-terminus, junction site (unique to APP-695) insert sequence (unique to APP-751,-770), A4 region, and C-terminus of APP to immunolabel sections from throughout the young adult rat brain. From these results we constructed maps of the staining pattern of each antibody. We found that APP is widely distributed throughout the brain, that labelling is predominantly neuronal in character, and that there is marked variation among the antibodies in the extent of labelling, the particular cell populations stained, and the structures labelled within individual cells. The differential staining patterns observed with the five different antibodies suggest that the way APP is processed differs from one region to another and within different compartments in the cell. The specificity of the antibodies was established by Western blot analysis, in which APP species of approximately 95 and 110 kD were found. Our findings on the distribution of APP provide a foundation for further investigations into the normal role of APP and the pathogenesis of AD.

Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD).

cDNAs encoding human family-IV phosphodiesterase, subtype D (hPDE IVD) were isolated from a human heart cDNA library. The overlapping cDNAs encode a polypeptide of 604 amino acids (aa) with a predicted M(r) of 68,502, which is 91.4% identical to the rat homolog, rPDE IVD. hPDE IVD produced in Escherichia coli was inhibited by rolipram. Expression of the hPDE IVD mRNA is widespread in human tissues and most abundant in skeletal muscle.

Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease.

In cerebral amyloid angiopathy, the amyloid-beta (A beta) deposits lie primarily in the tunica media suggesting that smooth muscle cells play an important role in A beta deposition. To define this role, we conducted an immunocytochemical study of brain tissue from cases of Alzheimer disease with extensive cerebral amyloid angiopathy and cerebral hemorrhage. Antibodies specific to recombinant beta protein precursor (beta PP) and synthetic peptides homologous to various beta PP sequences from residue 18 to 689 of beta PP695 were used. Antibodies to actin, tropomyosin, alpha-actinin or desmin were used to label muscle cells. Antibodies to A beta sequences intensely recognized the extracellular amyloid deposit. Antibodies raised against beta PP sequences other than the A beta domain recognized smooth muscle cells. beta PP-immunoreactivity was reduced in regions of A beta deposits, since no muscle cells were recognized by cytoskeletal markers or observed ultrastructurally. In order to assess why A beta is deposited in the tunica media, we used biotin-labelled beta PP to determine if beta PP can be locally retained. We found beta PP bound to the tunica media of vessels but not other brain elements. These findings suggest A beta in blood vessels derives from degenerating beta PP-containing smooth muscle cells.

The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family.

Five protein families are needed to encompass the diversity of cyclic-AMP (cAMP) phosphodiesterases (PDE). Family IV PDEs (PDE IV) specifically hydrolyze cAMP with a low Km, and are selectively inhibited by rolipram (Rp) and related drugs. Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D. Using RN from a human lymphocytic B-cell line (43D-Cl2), we have isolated a 3.8-kb cDNA by low-stringency screening using a rat PDE IV member B (r-PDE IVB) probe. Expression of the human (h) cDNA in Escherichia coli results in cAMP-specific PDE activity that is Rp sensitive. A single large open reading frame (ORF) predicts a 564-amino-acid protein with 92.9% identity to r-PDE IVB; at the nucleotide level the identity is 86.3%. This h-PDE IVB clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes [Livi et al., Mol. Cell. Biol. 10 (1990) 2678-2686]. Our analysis identifies the monocyte clone with r-PDE IVA. Southern blots using a 1.2-kb h-PDE IVB probe at low stringency suggest the presence of additional uncloned human PDE IV family members. Analysis of genomic Southern blots using short specific probes from the h-PDE IVA and h-PDE IVB cDNAs indicates that distinct genes encode these two PDE IV family members. RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IVA and 3.7 kb for h-PDE IVB.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of the beta amyloid precursor protein at sites of ischemic injury in rat brain.

We used various antibodies to the beta amyloid precursor protein (APP) of Alzheimer's disease to study changes in the cellular distribution of APP in experimental ischemic brain injury. In contrast to sham operated controls, rats with repeated reversible occlusions of one middle cerebral artery showed striking APP reactivity in astrocytic processes in perifocal regions and white matter tracts. Dystrophic axons and neurons with accumulated APP were also evident in the ipsilateral neocortex and hippocampus. Such changes were also apparent in rats subjected to partial forebrain ischemia by bilateral occlusion of the carotid arteries. Our studies suggest that focal ischemic insults or chronic hypoperfusion leads to increased accumulation of APP in surviving brain cells that may pertain to enhanced beta amyloid deposition in Alzheimer's disease.

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study.

Increasing recognition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimer's disease (AD) were examined immunohistochemically with antibodies to domain-specific sites of APP (N-terminal, C-terminal, beta A4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with beta A4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed only rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to beta A4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled only cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.

Expression cloning of a rat B2 bradykinin receptor.

A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8]-bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot high-stringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief.

Accumulation of amyloid precursor fragment in Alzheimer plaques.

Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimer's disease (AD). Amyloid beta/A4-protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N-terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule.

Characterization of beta-amyloid precursor proteins with or without the protease-inhibitor domain using anti-peptide antibodies.

Alternative splicing of the transcript encoding the beta-amyloid precursor protein (BAPP) of Alzheimer's disease produces multiple mRNA species. Translation of these mRNAs predicts protein products of 770, 751, and 695 amino acids. The difference arises from the inclusion in BAPP-770/751 of a 56-residue insert region which is homologous to Kunitz-type protease inhibitors. We have prepared and affinity-purified anti-peptide antibodies that react specifically with either BAPP-770/751 (insert-specific) or BAPP-695 (junction-specific). A detectable level of the mRNA corresponding to the BAPP-770/751 protein was found in all cell lines tested. Immunoprecipitation of 35S-labeled proteins from these cell lines showed them to contain one or two Mr 105,000 bands reactive with the insert-specific serum, i-291. In contrast, only cos-7 cells and the human neuroblastoma cell line, IMR-32, contained mRNA species that encode the BAPP-695 protein, as shown by Northern analysis with a junction-spanning oligonucleotide probe. A band of Mr 95,000 was immunoprecipitated specifically from these two cell lines using the junction-specific serum, J-284. Indirect immunofluorescence labeling of cells corroborated these findings. All cells reacted with the insert-specific antibodies, i-291 and i-324. Only cos-7 and IMR-32 cells reacted with the junction-specific antibody, J-284. These results demonstrate the usefulness of anti-peptide antibodies for the differential detection of the BAPP-695 and BAPP-770/751 proteins.

Processing of Alzheimer's disease-associated beta-amyloid precursor protein.

Studies were undertaken on the processing of Alzheimer's disease-associated beta-amyloid precursor protein in normal cultured human fibroblasts and a human neuroblastoma cell line. Major differences in processing between the secreted and intracellular forms of the precursor were found. The intracellular form appears to undergo amino-terminal processing yielding many smaller fragments, whereas the secreted form does not show any further proteolytic cleavage after its release from the cell surface. In pulse-chase experiments, antibodies to the A4 region immunoprecipitated bands of Mr = 92,000-128,000, which represent the intact precursor; several smaller intracellular fragments of Mr = 70,000-72,000, 55,000, 33,000 and 6,000 also immunoprecipitated with this antibody. The Mr = 6,000 band cleared from the cell very quickly and is postulated to be the A4-carrying remnant of the secreted protein. The data show that a fragment of Mr = 33,000, which includes the A4 region, is one stable processed end-product of the intracellular precursor protein. It is possible that different posttranslational modifications are the signals responsible for the differences in processing between the secreted and intracellular amyloid precursor protein.

Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein.

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.

Multiple forms and cellular localization of Drosophila DNA topoisomerase II.

Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073-1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself.

DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization.

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.